Genetic transformation of plants has been one of the major advances achieved in biotechnology and its contributions to producing improved plants, improved crops, and consequently improved availability of food worldwide has been widely recognized. In certain plants, however, transformation has been especially difficult to achieve, and transformation of the valuable forage crop alfalfa, Medicago sativa has been inhibited by the peculiarities of the plant.
Transformation of alfalfa has been hampered primarily by two major limitations: constraints imposed by the method of transformation, and the poor regeneration from tissue and cell cultures of many alfalfa varieties.
The first limitation occurs because alfalfa is presently primarily transformed through the use of Agrobacterium tumefaciens. Agrobacterium exhibits host strain specificity and only certain Agrobacterium strains will infect a few alfalfa genotypes. The ability to transform alfalfa is considerably limited as a result. The second major inhibition of transformation of alfalfa is its own poor regeneration frequency. Only a few varieties exhibit even modest regeneration, and those elite varieties providing superior performance in the field are notoriously poor regenerators. The combination of these two problems has created a considerable bottle-neck in achieving transformation of the plant.
Alfalfa exhibits other traits setting it apart from many crop plants. It is an autotetraploid and is frequently self incompatible in breeding. When selfed, the pollen may not germinate or, when it does, later stops germinating. Thus producing a true breeding parent for hybrids is not possible, which complicates breeding substantially. It has been determined that there are nine major germ-plasma sources of alfalfa: M. falcata, Ladak, M. varia, Turkistan, Flemish, Chilean, Peruvian, Indian, and African.
Culture of explant source tissue, such as mature cotyledons and hypocotyls, demonstrates the regeneration frequency of genotypes in most cultivars is only about ten percent. SeitzKris, M. H. and E. T. Bingham, In vitro Cellular and Developmental Biology 24 (10): 1047-1052 (1988). Efforts have been underway to improve regeneration, and have included attempts at asexual propagation to maintain individual genotypes which possess the regeneration trait. Further, propagation by asexual methods is not practical if many genotypes are involved. Bingham and others have attempted to avoid this problem by recurrent selection. In the first cycle, regenerating genotypes were selected, crossed and recycled until regeneration was improved to 60 percent or better. The result of this was the development of Regen-S, in which two-thirds of the plants were capable of regeneration from callus tissue. E. T. Bingham, et. al., Crop Science 15:719-721 (1975).
Additionally, researchers believe that somatic embryogenesis in alfalfa is inheritable, and is controlled by relatively few genes. Efforts at improving regeneration have thus been directed towards isolation of the genetic control of embryogenesis, and breeding programs which would incorporate such information. See, e.g. M. M. Hernandez-Fernandez, and B. R. Christie, Genome 32:318-321 (1989); I. M. Ray and E. T. Bingham, Science 29:1545-1548 (1989). This is complicated by the characteristics of alfalfa noted above.
This invention relates to improvements in transformation and regeneration of alfalfa by departing from these previous approaches. In one embodiment, direct introduction of DNA is accomplished by the use of microprojectile bombardment. As a result of the use of bombardment, the limitations of Agrobacterium are overcome.
Furthermore, limitations in regeneration of alfalfa are overcome by selecting immature cotyledons for transformation and regeneration. It has been found that when immature cotyledons of alfalfa are used, regeneration is considerably improved, and there are no limitations on what type of alfalfa can be regenerated as a result of this method. Thus even elite varieties may be regenerated, and transformed and there is no longer a limitation imposed by the method of transformation used, or variety transformed.
Thus, it is an object of this invention to improve transformation rates of Medicago sativa. 
It is another object of this invention to improve regeneration of Medicago sativa. A still further object of this invention is to allow transformation and regeneration of any variety of Medicago sativa. 
Still further objects of the invention will become apparent through the following description.
Microprojectile bombardment is used to transform DNA into Medicago sativa, resulting in introduction of DNA into any variety of Medicago sativa. The invention further relates to the use of immature cotyledons of Medicago sativa for transformation and regeneration of any variety of Medicago sativa.